Physiologically active substances

ABSTRACT

Physiologically active substances ML-236 of the formula ##SPC1## 
     Wherein R is hydrogen atom, hydroxy group or 2-methylbutyryloxy group having cholesterol- and lipid-lowering effects in blood and liver and thus utility as hypocholesteremic and hypolipemic medicaments. They are obtained by cultivation of an ML-236-producing microorganism belonging to the genus Penicillium in a culture medium and subsequent recovery thereof from a cultured broth.

This invention relates to new physiologically active substances and afermentative process for producing the same.

More particularly, it is concerned with a new class of physiologicallyactive substances (hereinafter referred to as ML-236) having the formula##SPC2##

Wherein R is hydrogen atom, hydroxy group or 2-methylbutyryloxy group(--OCOCH(CH₃)CH₂ CH₃) and also with a process for the production of thesubstances ML-236 by cultivation of an ML-236-producing microorganismbelonging to the genus Penicillium.

The new substances ML-236 of this invention have excellent physiologicalactivities for medicinol use, more specifically, an inhibition activityof cholesterol biosynthesis, an antiatherosclerosis activity and anantihyperlipemia activity.

One cause of such diseases as atherosclerosis, hyperlipemia and so on isnow considered to be owing to cholesterol deposit within a living body,especially in the intima of arteries.

Under these circumstances, we have made systematic studies on substancesobtainable from cultured broth of microorganisms about their inhibitionactivities of cholesterol biosynthesis, from a standpoint whereininhibition of cholesterol biosynthesis may be effective in preventingand treating such diseases and, as a result, it has been found that thesubstances ML-236 can be isolated from a cultured broth of amicroorganism belonging to the genus Penicillium and also that thesubstances ML-236 possess potent cholesterol- and lipid-lowering effectsin blood and liver.

It is, accordingly, a primary object of this invention to provide a newgroup of the substances ML-236 of the above formula (I) which are highlyuseful as hypocholesteremic agents and hypolipemic agents for thetreatment of atherosclerosis, hyperlipemia and like diseases.

Another object of this invention is to provide a process for thefermentative production of the valuable substance ML-236 by cultivationof an ML-236-producing microorganism belonging to the genus Penicillium.

Other objects and advantages of this invention will be apparent from thedescription as shown hereunder.

In one aspect of this invention, there are provided the substancesML-236 of the formula (I) as briefly explained hereinabove. It has nowbeen clarified that the substances ML-236 of the formula (I) areseparated into 3 kinds of substances which are designated as ML-236A,ML-236B and ML-236C, respectively, and the respective plane structuresthereof have been clarified as defined below, with the NMR, mass, IR,and UV spectra and X-ray diffractiometries thereof. ##SPC3##

The physico-chemical properties of the substances ML-236A, ML-236B andML-236C are summarized as shown below.

    ______________________________________                                                    ML-236                                                                          A         B          C                                                        oily      white scales                                                                             oily                                       Nature        (neutral) (neutral)  (neutral)                                  ______________________________________                                        m.p.              --        149-151°C.                                                                      --                                       Elementary                                                                             C        70.18     70.68    74.04                                    Analysis (%)                                                                           H         8.75      8.56     8.58                                             O        21.07     20.76    17.38                                    Molecular Weight                                                                            306        390        290                                       (Mass spectrum)                                                               Molecular Formula                                                                           C.sub.18 H.sub.26 O.sub.4                                                                C.sub.23 H.sub.34 O.sub.5                                                                C.sub.18 H.sub.26 O.sub.3                 UV spectrum   As shown   As shown   As shown                                  (Methanol)    in FIG. 1  in FIG. 3  in FIG. 5                                 IR spectrum   As shown   As shown   As shown                                  (KBr)         in FIG. 2  in FIG. 4  in FIG. 6                                 Solubility  Soluble in methanol, ethanol, acetone,                                        ethyl acetate, chloroform and benzene                                         Insoluble in n-hexane and petroleum                                           ether                                                             R.sub.f value                                                                        n-hexane-                                                                     acetone    0.21       0.46     0.52                                    (TLC : (1:1)                                                                  Silica                                                                        gel G)                                                                               dichloro-                                                                     methane-                                                                      ethyl      0.08       0.21     0.27                                           acetate                                                                       (7:3)                                                                  ______________________________________                                    

In FIGS. 1 through 6 hereof, FIG. 1 shows the ultraviolet absorptionspectrum of ML-236A exhibiting maxima at 229, 236 and 245 mμ,respectively; and FIG. 2 shows the infrared absorption spectrum ofML-236A showing absorption bands at 3350, 3300 and 1725 cm⁻ ¹,respectively.

FIG. 3 shows the ultraviolet absorption spectrum of ML-236B exhibitingmaxima at 229, 236 and 245 mμ, respectively; and FIG. 4 shows theinfrared absorption spectrum of ML-236B showing absorption bands at3500, 1740 and 1695 cm⁻ ¹, respectively.

FIG. 5 shows the ultraviolet absorption spectrum of ML-236C exhibitingmaxima at 229, 236 and 245 mμ, respectively; and FIG. 6 shows theinfrared absorption spectrum of ML-236C showing absorption bands at 3350and 1700 cm⁻ ¹, respectively.

The substances ML-236 of this invention, when applied to patientssuffering from such diseases as atheroscrelosis, hyperlipemia and so on,may be administered orally or parenterally in the form of a capsule, atablet, an injectable preparation and the like and it is usuallydesirable to administer the substance via oral route. Doses may bevaried depending upon the age, severity, body weight and otherconditions of patients, but usual daily dosage for adult is within arange from about 200 mg. to 2000 mg. given in 3 or 4 divided doses.However, higher doses may be favorably applied, as required.

As fully disclosed hereinabove, the substances ML-236 of this inventionhave potent activities for the inhibition of cholesterol biosynthesis invitro and in vivo.

I. The activity in vitro on inhibition of cholesterol biosynthesis ofthe substances ML-236 may be assayed by the following method. Slice ofrat liver and radioactive acetic acid are brought to interaction at37°C. for 60 minutes, the radioactive cholesterol thus biosynthesized issaponified and separated as precipitates with digitonin and then itsradioactivity is measured to determine the produced cholesterol amount.Separately, the same procedures as above are employed except that ML-236is added at the beginning of the reaction, thereby the biosynthesizedcholesterol amount being determined. Thus, an effect of ML-236 can bequantitatively determined. (See Bricker et al.: The Journal ofBiological Chemistry, 247, 4914, 1972)

According to the assay method as set forth above, it has been shown thatthe substances ML-236A, ML-236B and ML-236C exhibit about 50% inhibitionof cholesterol biosynthesis at the respective concentrations of 0.04μg./ml., 0.01 μg./ml. and 0.08 μg./ml. Further, acute toxicity of eachsubstance ML-236 was determined in mice via intraperitonealadministration to be 400 mg./kg. or more, which clearly shows a lowertoxicity of the substance ML-236.

II. Effectiveness of ML-236 has been confirmed by examining its effecton lowering the lipid content in blood and liver of animals with varioustest methods, representatives of which will be more fully illustratedbelow.

1. One of two groups of rats having an average weight of about 200 g.,each group consisting of 5 animals, was individually given byintravenous injection with 200 mg./kg. of Triton WR 1339 (trade name;available from Ruger Chemical Co., Ltd., U.S.A.; having an activity ofincreasing cholesterol level in blood) simultaneously withintraperitoneal administration of 20 mg./kg. of ML-236B. After 24 hoursfrom administration, rats were sacrificed by bleeding, blood and liverwere collected and their cholesterol and neutral lipid levels weredetermined by a conventional method. As a result, it has been clear thatcholesterol levels in blood are lowered by 14.2% and those in liver by10.1%, as compared with those in the case of another group given withintravenous injection of Triton WR 1339 alone.

2. Two of four groups of rats, each group consisting of five animals,were orally given one dose of 5 mg./kg. of ML-236A suspended in gumarabic. After 3 hours and 18 hours from administration, animals weresacrificed by bleeding and blood was collected. Cholesterol and neutrallipid levels in blood serum were determined by a conventional method. Ithas been clear that the cholesterol and neutral lipid levels after 3hours are lowered by 13.5% and 49.5%, respectively, and also that thecholesterol level after 18 hours is lowered by 13.5%, while lowering ofneutral lipid level is not observed, as compared with those in the caseof two other groups administered with gum arabic alone.

Moreover, one of two other groups of rats, each group consisting of fiveanimals, were orally given 80 mg./kg. of powdery ML-236B in the form ofa physiological saline solution. After 3 hours from administration,cholesterol and neutral lipid levels in blood serum were determined inthe same manner as above. It has been clear that the cholesterol andneutral lipid levels are lowered by 20.0% and 44.2%, respectively, ascompared with those in the case of another group administered with aphysiological saline solution containing no ML-236B.

It will be apparent from the above results that the substances ML-236 ofthis invention exhibit a prominent effect in the inhibition ofcholesterol biosynthesis in a living body and thus they are utilizableas a medicament against, for example, atherosclerosis, hyperlipemia andso on.

In another aspect of this invention, there is provided a process forproducing physiologically active substances ML-236A, ML-236B and ML-236Cwhich comprises cultivating an ML-236-producing microorganism belongingto the genus Penicillium and then recovering said substances from acultured broth.

The microorganisms which may be employed in this invention are theML-236-producing ones belonging to the genus Penicillium and, as thestrain proved to be particularly effective for this invention, there is,for instance, mentioned Penicillium citrinum SANK 18767 which has beendeposited under an accession No. 2609 with Technical Research Instituteof Microbial Industry, Agency of Industrial Science & Technology,Ministry of International Trade and Industry, Japan, and also asNRRL-8082 in the Northern Regional Research Laboratory, Northern CentralRegion, Agricultural Research Service, U.S. Department of Agriculture,at Peoria, Ill., U.S.A.

Although this invention will be explained hereinbelow principally withrespect to the strain SANK 18767, it is well-known in the art thatvarious properties of all microorganisms belonging to the genusPenicillium are not definite, but the microorganisms of the genusPenicillium may be easily varied naturally and artificially. It is,accordingly, to be noted that all strains which are of the genusPencillium and capable of producing ML-236, including varieties andmutants, are contemplated and usable in this invention.

The above strain SANK 18767 itself is known and its morphologicalproperties are reported in the following literature: K. B. Raper and C.Thom; A Manual of the Penicillia, the Williams and Wilkins Company,1949.

In carrying out the process of this invention, cultivation may besatisfactorily conducted under aerobic condition in the same manner ascommonly employed in the art for cultivation of a strain of the genusPenicillium. For instance, the ML-236-producing microorganism may begrown in a culture medium, e.g., that containing malt extract 2%,glucose 2%, peptone 1%, agar 2% and then the culture so obtained may beinoculated and cultivated in a culture medium. Alternatively, themicroorganism grown in a culture medium may be cultivated in anotherfresh culture medium to produce ML-236.

As medium components may be employed any of the well-known nutrientmaterials for Penicillium. For instance, as an assimilable carbonsource, glucose, glycerol, maltose, dextrin, starch, lactose, sucrose,molasses, soybean oil, cotton seed oil, etc., preferably glucose andmaltose may be employed and, as an assimilable nitrogen source, soybeanmeal, peanut meal, cotton seed meal, fish meal, corn steep liquor,peptone, rice bran, meat extract, yeast, yeast extract, sodium nitrate,ammonium nitrate, ammonium sulfate, etc. may be used. And, suchinorganic salts as sodium chloride, phosphates, calcium carbonates,etc., may be added to a culture medium. A minor amount of a metal saltmay also be added, if necessary. Further, a minor amount of a heavymetal may be added, if necessary.

Particularly, in cultivating the ML-236-producing microorganism underaerobic condition, ordinary aerobic cultivation methods such as, forexample, solid culture, culture under aeration and agitation, shakenculture, etc., may be favorably utilized.

In carrying out cultivation with aeration and agitation, an anti-foamingagent, e.g., silicon oil, vegetable oils, surfactants, etc., may besuitably employed.

The pH of the medium may be usually within a pH range of 3-9 andpreferably within or around neutral range and cultivation temperaturemay be usually of 20°-30°C., in particular about 28°C. being preferred.

Cultivation may be continued until ML-236 will be substantiallyaccumulated in a culture medium, usually for 20 hours to 240 hours,preferably for 48 hours to 168 hours and, after cultivation, each ML-236substance may be isolated and recovered from a cultured broth by asuitable combination of various methods related to the propertiesthereof which has been elucidated by us, as illustrated in the examplesgiven below. For example, there may be mentioned extraction with anorganic solvent, e.g., ether, ethyl acetate or chloroform; dissolutioninto a more polar solvent, e.g., acetone or alcohol; removal ofimpurities with a less polar solvent, e.g., petroleum ether or hexane;adsorptive chromatography with active carbon or silica gel; gelfiltration through a column of "Sephadex" (trade name; available fromPharmacia Co., Ltd., U.S.A.); and so on. By the use of a suitablecombination of these measures the crystalline or oily form of thepresent substance can be isolated from a cultured broth.

The present invention will be more fully illustrated by way of thefollowing examples, but they are not intended to limit the scope of thisinvention. Various modifications of the present process may be made bythose skilled in the art, in particular, for the recovery of ML-236A,ML-236B and ML-236C from a cultured broth, in view of the above-depictedproperties.

EXAMPLE 1

Into a 600 l.-volume fermenter was charged 300 l. of a culture mediumcontaining glucose 2%, peptone ("Kyokuto," trade name; available fromKyokuto Seiyaku K.K., Japan) 0.1% and malt extract 3% and Penicilliumcitrinum SANK 18767 was inoculated thereon. Cultivation was conducted ata temperature of 28°C., an aeration of 300 l./min. and an agitation of190 r.p.m. for 91 hours.

The cultured broth was filtered by a filter press to give 280 l. of thefiltrate. The filtrate so obtained was adjusted to pH 4.0 with 6Nhydrochloric acid and extracted twice with 250 l. of ethyl acetate.

The extract was concentrated to dryness under reduced pressure to give150 g. of the dried product. 120 g. of the product was adsorbed on acolumn of 1040 g. of silica gel ("Wakogel C-200"; trade name; WakoJunyaku K.K., Japan), which was developed in turn with 5 l. of benzene,7.5 l. of benzene-ethyl acetate (95:5) and then 28 l. of benzene-ethylacetate (80:20). Active eluates are of two fractions, namely the firsteluted fraction named C (containing ML-236C) and the second elutedfraction named B (containing ML-236B). Further, the column was developedwith 2 l. of acetone to give the third fraction named A (containingML-236A). The former two fractions were concentrated to dryness so that2.42 g. and 2.61 g. of the dried products were obtained from thefractions C and B, respectively.

The fraction B (2.61 g.) was dissolved in 100 ml. of benzene, theresulting solution was washed with 100 ml. of water and the washingswere discarded. The benzene layer was dried over sodium sulfate andconcentrated and the residue was allowed to stand overnight, whereuponthe so separated crystalline substance was recovered. The substance wasthen recrystallized once from benzene and subsequently once from ethanolto give 232 mg. of ML-236B in a pure state as white crystals. Thissubstance was confirmed as pure by a thin-layer chromatography and showsan inhibitory activity of about 50% on biosynthesis of cholesterol in aconcentration of 0.01 μg./ml.

EXAMPLE 2

Into a 6 ton-volume fermenter was charged 3000 l. of the same culturemedium as in Example 1 and Penicillium citrinum SANK 18767 wasinoculated thereon. Cultivation was conducted for 90 hours in the samemanner as in Example 1. The cultured broth was filtered by a filterpress to give 2590 l. of the filtrate. The filtrate was concentratedunder reduced pressure to 450 l. and the concentrate was adjusted to pH4.0 with 6N hydrochloric acid and then extracted with 450 l. of ethylacetate. 390 l. of the extract was concentrated to dryness to yield 346g. of an oily substance. The substance was adsorbed on a column of 5.18kg. of silica gel ("Wakogel C-200"), which was then developed in turnwith 98 l. of n-hexane, 60 l. of n-hexane -acetone (95:5) and then 150l. of n-hexane-acetone (85:15). The resulting active fraction (42 1.;containing ML-236B and other active substances) was concentrated todryness to leave 40 g. of the dried product. This product was dissolvedin a suitable amount of benzene and insolubles were filtered off. Thefiltrate was left overnight, whereupon ML-236B was separated out ascrystals and other active substances were retained in a waste solution.The crystals were collected by filtration and then recrystallized oncefrom benzene and subsequently once from ethanol to obtain 12.8 g. of thepure ML-236B.

EXAMPLE 3

Into a 6 ton-volume fermenter was charged 3000 l. of a culture mediumcontaining glucose 2%, peptone (Kyokuto) 0.1% and malt extract 3% andPenicillium citrinum SANK 18767 was inoculated thereon. Cultivation wasconducted at a temperature of 28°C., an aeration of 3000 l./min. and anagitation of 145 r.p.m. for 96 hours.

The cultured broth was filtered by a filter press to give 2900 l. of thefiltrate. The filtrate so obtained was concentrated under reducedpressure to 450 l. and the concentrate was then adjusted to pH 4.0 with6N hydrochloric acid followed by extraction with 500 l. of ethylacetate. 500 l. of the extract was concentrated to dryness to leave 327g. of an oily substance. The substance was adsorbed on a column of 5.5kg. of silica gel (Wakogel C-200), which was then developed in turn with10 l. of n-hexane, 60 l. of n-hexane-acetone (95:5) and then 150 l. ofn-hexane-acetone (85:15). Two active fractions were given, namely, thefirst eluted fraction containing ML-236C and the second eluted fractioncontaining ML-236B.

Thereafter, the column was developed with 20 l. of acetone, therebyeluting the third active fraction containing ML-236A.

Each respective fraction was individually concentrated to dryness.

The dried product (194 g.) from the ML-236A fraction was dissolved in 2l. of ethyl acetate and the resulting solution was extracted three timeswith 500 ml. portion of a saturated aqueous solution of sodiumcarbonate. The ethyl acetate layer was separated, further washed with asaturated aqueous solution of sodium chloride, dried over anhydroussodium sulfate and then concentrated to dryness to leave 70 g. of thedried product. The product was adsorbed on a column of 150 g. of silicagel (Wakogel C-200), which was then washed with 1 l. of benzene and 3 l.of benzene-ethyl acetate (8:2) and eluted with 5 l. of benzene-methanol(95:5). Main fractions were concentrated to dryness to give 9 g. ofML-236A as an oily substance.

The dried product (38 g.) from the ML-236B fraction was added to 500 ml.of benzene and insolubles were removed by filtration. The filtrate wasconcentrated and the concentrate was allowed to stand overnight,whereupon ML-236B was separated out as crystals. The so separatedcrystals were recovered by filtration and crystallized once from benzeneand subsequently once from ethanol to give 10.5 g. of ML-236B in a purestate.

Finally, the dried product (3.2 g.) from the ML-236C fraction wasdissolved in a small amount of dichloromethane and the resultingsolution was adsorbed on a column of 50 g. of silica gel (WakogelC-200), which was then developed in turn with 500 ml. of dichloromethaneand then dichloromethane-ethyl acetate (95:5). Fractions containingML-236C were collected and concentrated to dryness to give 2.1 g. ofML-236C as an oily substance.

What is claimed is:
 1. A compound having the formula ##SPC4##wherein Ris hydrogen atom, hydroxy group or 2-methylbutyryloxy group.
 2. Acompound according to claim 1 wherein said R is hydroxy group.
 3. Acompound according to claim 1 wherein said R is 2-methylbutyryloxygroup.
 4. A compound according to claim 1 wherein said R is hydrogenatom.